The present invention generally relates to the purification and/or characterization of libraries of compounds, for example, combinatorial and lead generation libraries.
Currently, there are many general methods for purifying synthetic compounds. These methods generally involve purifying a single target compound from multiple impurities.
Compounds are currently being prepared in relatively large numbers in combinatorial and lead generation libraries. Often, compounds are synthesized in multi-well plates or multi-tube arrays, with the number of related compounds numbering in the thousands. It is difficult to purify and characterize the libraries of compounds at the rate at which they are synthesized.
One method for purifying large numbers of compounds involves a repetitive method of chromatographing individual compounds. This constitutes a full cycle of synthesis, work-up, and purification for each molecule. Often, a large amount of time is spent developing an appropriate purification method for the compounds to be purified.
It would be advantageous to develop efficient methods for purifying and/or characterizing libraries of compounds which are applicable to a wide variety of organic compounds. The present invention provides such methods.
Methods for purifying and/or characterizing compounds, particularly libraries of compounds such as combinatorial or lead generation libraries, are disclosed. Purification devices capable of being used in the method are also disclosed.
The method is premised on the discovery that structurally similar compounds, such as those in combinatorial and lead generation libraries, often have roughly similar retention factors (Rfs) on thin layer chromatography (TLC) plates and retention times on high performance liquid chromatography (HPLC) columns. The method uses this discovery to determine optimum conditions for purifying a library of similar compounds.
For a given column packing, solvent system, and flow rate, most compounds tend to elute to a certain degree from an analytical and/or preparative HPLC column. Similarly, most compounds move to a certain degree on TLC plates. While some compounds do not move at all (Rf=0), others move with a low Rf (for example, 0.05 less than Rf less than 0.2), a medium Rf (for example, 0.2 less than Rf less than 0.8), or a high Rf (for example, Rf greater than 0.8). One can readily determine a series of three or more, preferably four or more, zones of Rfs, or, in the case of analytical HPLCs, a series of zones of retention times, in which the majority of compounds in the library will elute (in the case of an analytical HPLC) or move (in the case of a TLC), for example, low, medium and high Rfs on a TLC plate. These zones can be correlated to preparative or semi-preparative methods for performing preparative or semi-preparative HPLCs.
For a given analytical HPLC and/or TLC protocol, a set of preparative HPLC conditions can be identified wherein compounds in one zone can be separated from compounds in other zones. Accordingly, if all or substantially all of the compounds in a representative sample of a library of compounds is present in the same zone, the library can be purified using the same HPLC protocol, which can be readily correlated to a zone on the TLC and/or analytical HPLC.
The methods described herein involve evaluating a representative sample of compounds from a library of compounds, such as a combinatorial or lead generation library, by TLC and/or analytical HPLC, to determine which zone they move on the TLC plate and/or elute off the analytical HPLC column. Once the zone is identified, a correlated preparative or semi-preparative method is used to purify the library.
Appropriate conditions for purifying a library of compounds can be worked out by route scouting a representative sample of the library for a given analytical HPLC column, solvent system and flow rate, and/or a given TLC backing and solvent system. A correlated preparative or semi-preparative HPLC method can be applied to purify the library of compounds without having to change the purification parameters, so that a single method can be applied to the entire library.
A suitable sample size is typically on the order of between 2 and 5% of the library, depending on the diversity of the compounds in the library. This approach is referred to herein as xe2x80x9croute scouting,xe2x80x9d since one is scouting for an appropriate purification route.
Preferably, both the TLC and the HPLC are performed on the representative sample of compounds. It is also preferred to perform preparative or semi-preparative HPLC on a sample of compounds from the library before purifying the entire library. This allows one to verify that the conditions are suitable for purifying the entire library, for example, by determining the purity of the compounds in the representative sample. Also, one can perform a TLC on between 10 and 100%, preferably between 50 and 100% of the library and compare the TLC to that in the representative sample. By performing a TLC on the entire library and/or determining the purity of a representative sample of compounds, one can ensure that the majority of the library can be adequately purified. If the purity is not adequate on the representative sample, or if the TLC of the library does not sufficiently match that of the representative sample, alternative preparative HPLC conditions can be used.
The representative sample should encompass, if possible, the most polar and least polar compounds synthesized in the library, to help ensure that the method is applicable for the entire library.
The method described herein results in substantial time savings in the purification and characterization of libraries of compounds, and can provide compounds with greater than 90% purity.